This year cancer will become the leading cause of death worldwide. The global burden of cancer doubled between 1975 and 2000. By 2020, it is projected to double again, and to triple by 2030. Ideally, any cancer therapy should be effective (at killing cancerous cells), targeted (i.e. selective, to avoid killing healthy cells), permanent (to avoid relapse and metastasis), and affordable. The ultimate goal of this work is to meet each of these criteria. The studies described build upon a significant body of work on a chimeric NKG2D (chNKG2D) receptor modified T cell therapy. This process will be made into a product through a biotech innovation. This innovation will be applicable to most other targeted cell-based therapies. Allogeneic immune cells will attack the host, causing graft-versus-host disease. Allogeneic T cells mediate this response through the T cell receptor (TCR). We have designed, and will create and analyze a T cell which expresses a targeting receptor but does not express TCR. For any non-TCR-based targeting strategy (e.g. cell based Fv-targeting), this will permit allogeneic therapies, thereby transforming a set of process-based therapies (which rely on autologous cells) to product-based therapies (which could be manufactured at a single location using cells from healthy donors). This distinction is critical to both cost and quality control. The chNKG2D receptor is attractive as it kills tumor cells and significantly alters the tumor microenvironment with excellent specificity. Further, the therapy activates host immunity against other tumor antigens, which broadens targeting and will make it less likely for tumor variants to emerge. Finally, the modified T cells are quickly eliminated from the host, but the induced immune response is durable and protects against future tumor challenge, even in the absence of the modified T cells. The chNKG2D approach is also compelling in that it is not limited to a single cancer indication. In fact, 70% of all cancers may express the ligands for this receptor. In vivo data in murine myeloma, lymphoma, and ovarian cancer models, has demonstrated safety and efficacy against both liquid and solid tumors. ChNKG2D-targeted T cells from allogeneic sources would be the first example of a wholly new paradigm for cellular cancer therapies. We will demonstrate the ability to remove TCR from primary T cells using shRNA targeted to TCR-(, TCR-(, and CD3( chains (Aim I). We will then demonstrate the combined expression of our targeting receptor and loss of TCR in primary human T cells (Aim II), yielding a promising therapeutic platform, ready for preclinical development. PUBLIC HEALTH RELEVANCE: We have been able to target and kill both liquid and solid cancers, and to simultaneously train the patient's own immune system to do likewise (e.g. to prevent metastatic disease) through the use of modified immune cells from individual patients. This project aims to transition this process into a product, thereby dramatically decreasing cost and improving process control by enabling the use of cells from healthy donors.